Interferon-alpha activates multiple STAT proteins and upregulates proliferation-associated IL-2Ralpha, c-myc, and pim-1 genes in human T cells

Interferon-alpha (IFN-alpha) is a pleiotropic cytokine that has antiviral, antiproliferative, and immunoregulatory functions. There is increasing evidence that IFN-alpha has an important role in T-cell biology. We have analyzed the expression of IL-2Ralpha, c-myc, and pim-1 genes in anti-CD3-activated human T lymphocytes. The induction of these genes is associated with interleukin-2 (IL-2)-induced T-cell proliferation. Treatment of T lymphocytes with IFN-alpha, IL-2, IL-12, and IL-15 upregulated IL-2Ralpha, c-myc, and pim-1 gene expression. IFN-alpha also sensitized T cells to IL-2-induced proliferation, further suggesting that IFN-alpha may be involved in the regulation of T-cell mitogenesis. When we analyzed the nature of STAT proteins capable of binding to IL-2Ralpha, pim-1, and IRF-1 GAS elements after cytokine stimulation, we observed IFN-alpha-induced binding of STAT1, STAT3, and STAT4, but not STAT5 to all of these elements. Yet, IFN-alpha was able to activate binding of STAT5 to the high-affinity IFP53 GAS site. IFN-alpha enhanced tyrosine phosphorylation of STAT1, STAT3, STAT4, STAT5a, and STAT5b. IL-12 induced STAT4 and IL-2 and IL-15 induced STAT5 binding to the GAS elements. Taken together, our results suggest that IFN-alpha, IL-2, IL-12, and IL-15 have overlapping activities on human T cells. These findings thus emphasize the importance of IFN-alpha as a T-cell regulatory cytokine.     

Promegapoietin, a family of chimeric growth factors, supports megakaryocyte development through activation of IL-3 and c-Mpl ligand signaling pathways

OBJECTIVE: The signaling pathways induced by promegapoietin (PMP), a family of chimeric growth factors that activate the human IL-3 and c-Mpl receptors, were investigated. METHODS: The biological activity of PMP was examined by receptor binding, cell proliferation, ex vivo expansion of hematopoietic progenitor cells, and in vivo production of platelets. The activation of signaling pathways was examined by Western blot and Northern blot analyses. RESULTS: Two PMP molecules, PMP-1 and PMP-1a, induced proliferation of cells expressing the IL-3 receptor, c-Mpl, or both receptors and bound to the IL-3 receptor and c-Mpl with high affinity. Ex vivo expansion assays using human bone marrow CD34(+) cells suggested that PMP-1 induced greater total cellular expansion as well as expansion of CD41(+) megakaryocytic precursor cells than IL-3 or c-Mpl ligand alone. Subcutaneous administration of 50 microg/kg of PMP-1 for 10 days to rhesus monkeys resulted in increased platelet production in vivo from a baseline of 357 +/- 45 x 10(3) cells/mL to 1376 +/- 151 x 10(3) cell/mL. PMP-1 induced phosphorylation of the beta(c) subunit of IL-3 receptor and c-Mpl, JAK2, and STAT5b, but not STAT3. PMP-1 induced greater expression of Pim-1, c-Myc, and cyclin D2 than did either an IL-3 receptor agonist or c-Mpl receptor agonist alone. The magnitude of induction of early response genes was similar for PMP and the coaddition of IL-3 receptor agonist and c-Mpl receptor agonist. CONCLUSION: PMP combines the biological activities of IL-3 and c-Mpl ligand in a single molecule that can simultaneously activate signaling pathways induced by both these ligands.     

Inhaled carbon monoxide suppresses the development of postoperative ileus in the murine small intestine

BACKGROUND & AIMS: The induction of heme oxygenase (HO-1), the rate-limiting enzyme in heme metabolism, is protective against injury in acute and chronic inflammation. Inhalation of low levels of carbon monoxide (CO), a byproduct of heme metabolism, has anti-inflammatory effects equal to HO-1 induction. This study examined whether inhaled CO was protective against the development of postoperative ileus. METHODS: Ileus was induced by surgical anesthesia and gentle manipulation of the mouse small intestine. Animals were exposed to CO (250 ppm) in air 1 hour before and continuously for 24 hours after surgery. RESULTS: CO inhalation prevented the manipulation-induced suppression of circular muscle contractility in vitro, and significantly improved gastrointestinal transit in vivo. Proinflammatory messenger RNA (mRNA) expression (interleukin [IL]-6, IL-1beta, cyclooxygenase 2 [COX-2], inducible nitric oxide [iNOS]) and anti-inflammatory mediator expression (IL-10 and HO-1) were elevated 3 to 6 hours after surgery relative to controls. CO treatment reduced IL-1beta and iNOS peak expression by 75%, but not IL-6 or COX-2. In manipulated mice treated with CO, HO-1 expression peaked earlier (3 hours after surgery) and at levels 300% higher than in mice not exposed to CO. IL-10 expression at 3 hours also was 300% higher after CO treatment. CONCLUSIONS: These findings suggest that CO attenuates postoperative ileus by inhibiting selective elements within the inflammatory cascade and by enhanced induction of the anti-inflammatory cytokine IL-10. In addition, the early and enhanced induction of HO-1 potentially amplifies the anti-inflammatory effects of the HO-1 pathway by protection from free radical stress and by increasing the tissue availability of CO directly at the sites of inflammation.